CN103175919B - Gas chromatography-mass spectrometry combined detection method for -NNAL and (R) -NNAL in urine - Google Patents
Gas chromatography-mass spectrometry combined detection method for -NNAL and (R) -NNAL in urine Download PDFInfo
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- 210000002700 urine Anatomy 0.000 title claims abstract description 67
- OGRXKBUCZFFSTL-SNVBAGLBSA-N n-[(4r)-4-hydroxy-4-pyridin-3-ylbutyl]-n-methylnitrous amide Chemical compound O=NN(C)CCC[C@@H](O)C1=CC=CN=C1 OGRXKBUCZFFSTL-SNVBAGLBSA-N 0.000 title claims abstract description 40
- 238000001514 detection method Methods 0.000 title claims abstract description 20
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 title abstract 2
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- LRHPLDYGYMQRHN-UHFFFAOYSA-N n-Butanol Substances CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- XKLJHFLUAHKGGU-UHFFFAOYSA-N nitrous amide Chemical compound ON=N XKLJHFLUAHKGGU-UHFFFAOYSA-N 0.000 description 2
- 238000002203 pretreatment Methods 0.000 description 2
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- KNPUXTWHFSLCDT-BBYIEOQPSA-N 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol glucuronide Chemical compound C=1C=CN=CC=1C(CCCN(C)N=O)O[C@@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O KNPUXTWHFSLCDT-BBYIEOQPSA-N 0.000 description 1
- OGRXKBUCZFFSTL-UHFFFAOYSA-N 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol Chemical compound O=NN(C)CCCC(O)C1=CC=CN=C1 OGRXKBUCZFFSTL-UHFFFAOYSA-N 0.000 description 1
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- 241000656145 Thyrsites atun Species 0.000 description 1
- AATFPUCRBIXXID-ROUUACIJSA-N [(2S)-3,3,3-trifluoro-2-methoxy-2-phenylpropanoyl] (2S)-3,3,3-trifluoro-2-methoxy-2-phenylpropanoate Chemical compound CO[C@](C(=O)OC([C@](OC)(C(F)(F)F)C1=CC=CC=C1)=O)(C1=CC=CC=C1)C(F)(F)F AATFPUCRBIXXID-ROUUACIJSA-N 0.000 description 1
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Abstract
A gas chromatography-mass spectrometry combined detection method of -NNAL and (R) -NNAL in urine, comprising: adding rac-NNAL-methyl-d 3 solution, ammonium dihydrogen phosphate buffer solution (pH6.4, 0.6M) and beta-glucuronidase into a urine sample, uniformly mixing, placing the mixture into a microwave-assisted extractor for enzymolysis at a constant temperature of 37 ℃, purifying the urine sample subjected to enzymolysis by using a MIP-NNAL solid phase extraction small column, concentrating the urine sample under reduced pressure until the urine sample is dry, adding triethylamine anhydrous acetonitrile solution and - (+) -alpha-methoxy-alpha-trifluoromethyl phenylacetyl chloride anhydrous acetonitrile solution, performing GC-MS-MS analysis on the sample after derivatization, and calculating the content of -NNAL and (R) -NNAL in the urine sample. The invention provides a method for analyzing and detecting -NNAL and (R) -NNAL in a urine sample, which has the advantages of low detection limit, good stability, simple pretreatment, rapidness and accuracy, and fills the blank in the technical field.
Description
Technical field
The invention belongs to the biomarker physical and chemical inspection technical field of tobacco-specific nitrosamine in urine, be specifically related to the gas chromatography-mass spectrum-mass spectrometry detection method of (S)-NNAL and (R)-NNAL in urine.
Background technology
In recent years, people more and more pay attention to smoking and health problem, therefore more and more deep to the research of the various harmful chemical components in flue gas both at home and abroad.Through research for many years, the toxicity assessment result of international cancer research institution (IARC) etc. is thought: 4-(methyl nitrosamino group)-1-(3-the pyridine radicals)-1-butanone (NNK) in tobacco-specific nitrosamine has carcinogenicity to animal, and NNK and its metabolin are potential lung cancer carcinogenic substances.Therefore, tobacco business is inside and outside in the urgent need to setting up the method for a kind of accurate evaluation different crowd to NNK degree of exposure now, and biomarker method is a kind of method of generally acknowledging and effectively monitoring NNK exposed population group degree of exposure.
Up to the present, numerous tobacco scientific workers' the proof that studies for a long period of time, in smoker's urine, NNK do not detected, the major metabolite of NNK in human body and laboratory animal body is 4-(methyl nitrosamino group)-1-(3-pyridine radicals)-n-butyl alcohol (NNAL) and NNAL glycoside compounds (comprising NNAL-O-Glucuronide and NNAL-N-Glucuronide), these metabolic products mainly excrete by urine and other body fluid.Although the NNK in tobacco and cigarette smoke has carcinogenicity, the toxicity of its main metabolites NNAL in human body significantly reduces, and after NNAL glucosidesization, toxicity further reduces.In biological metabolism sample analysis, because urine sample easily obtains, can effectively reflect again the exposure of NNK, so become study hotspot always.Now, detect NNAL in urine and NNAL glycoside compounds and become a kind of smoker of detection, smoke-free tobacco product consumer and involuntary smoker and be exposed to the conventional biomarker that contacts of NNK.
In NNK molecule, there is no asymmetric carbon atom, therefore NNK molecule does not have enantiomter, but in NNK molecule, there is a carbonyl, this carbonyl is along with NNK is reduced to hydroxyl by carbonyl reductase after entering human body, generate a pair of NNAL enantiomter, be respectively (S)-NNAL and (R)-NNAL.2002, the research of Hecht etc. is found to disclose, in people's urine sample, the content of (the R)-NNAL of free form is slightly approximately 1.1 times of (S)-NNAL content, but in people's urine sample the content of (R)-NNAL glycoside compounds be (S)-NNAL glycoside compounds content about twice, the further metabolic pathway difference of this explanation (S)-NNAL and (R)-NNAL is very large, body metabolism is discharged the speed of (S)-NNAL far below the speed of discharging (R)-NNAL, also illustrate that human body has a kind of stereoselective reserve capability to (S)-NNAL simultaneously, particularly the ratio of mouse significant period of time Nei Qi lung (S)-NNAL after absorption NNK is also far above (R)-NNAL.Other scholars' result of study also shows that in the patients with lung cancer urine sample of smoking, (S)-NNAL is obviously greater than common smoker with the ratio of (R)-NNAL.In conjunction with epidemiological survey and analysis result, people infer the acceptor that may have (S)-NNAL in people's lungs, and this acceptor can specific binding (S)-NNAL, and this may be exactly that smoking absorbs NNK and causes one of reason of lung cancer.
Because the NNK burst size in cigarette smoke only has every cigarette of several nanograms, and NNK is metabolised to many kinds of substance after entering human body, in addition urine sample matrix complexity, so (the S)-NNAL in urine sample and the detection of (R)-NNAL are very difficult, domestic and international many relevant industries all cannot be carried out the testing of (S)-NNAL and (R)-NNAL in urine sample.Under this background, many relevant industries in the urgent need to set up one can rapid and accurate determination human body urine sample in (S)-NNAL and the content of (R)-NNAL and the method for ratio, to provide with further reference to information for the early detection of lung cancer etc.For the deficiencies in the prior art, gas chromatography-mass spectrum-mass spectrometry detection method of (S)-NNAL and (R)-NNAL in urine provided by the invention, have that detectability is low, speed is fast, accuracy and feature highly sensitive, with low cost, be applicable to the detection of (S)-NNAL in the common sample of various human urines and (R)-NNAL, therefore the method is significant to the degree of exposure of NNK in flue gas for the content and the assessment human body that detect (S)-NNAL in urine and (R)-NNAL.
Summary of the invention
The object of the invention is to the deficiency that is difficult to carry out the testing of (S)-NNAL and (R)-NNAL in urine sample for existing method, gas chromatography-mass spectrum-mass spectrometry the detection method of (S)-NNAL and (R)-NNAL in a kind of urine is provided, for assessing the individual decision method that the degree of exposure of NNK in flue gas is provided to a kind of science, the method has that detectability is low, good stability, pre-treatment simply, the advantage such as quick and precisely.Gas chromatography-mass spectrum-mass spectrometry that this method is applicable to (S)-NNAL and (R)-NNAL in urine detects, and is the method for the degree of exposure of a kind of Scientific evaluation individuality to NNK in flue gas.
Object of the present invention is achieved by following technical proposals:
Gas chromatography-mass spectrum-mass spectrometry detection method of (S)-NNAL and (R)-NNAL in urine, comprises the following steps:
1. with the configuration of mensuration optically-active definite (the S)-NNAL of method comparing and (R)-NNAL and as reference, or with the configuration of definite (S)-NNAL-(S)-(+)-α-methoxyl-α-trifluoromethyl phenylacetic acid ester of Mosher method and (R)-NNAL-methyl-d3-(S)-(+)-α-methoxyl-α-trifluoromethyl phenylacetic acid ester and as reference;
2. the mixed standard solution that preparation contains rac-NNAL and deuterated rac-NNAL-methyl-d3; use again the anhydrous acetonitrile of (S)-(+)-α-methoxyl-α-trifluoromethyl phenyllacetyl chloride and the anhydrous acetonitrile derivatization of triethylamine, then the later mixed standard solution of derivatization is carried out to GC-MS-MS analysis; With determining that (S)-NNAL-(S)-(+)-α-methoxyl-α-trifluoromethyl phenylacetic acid ester and (R)-NNAL-(S)-(+)-α-methoxyl-α-trifluoromethyl phenylacetic acid ester after configuration determine corresponding chromatographic peak retention time by retention time, and set up the standard solution working curve of (S)-NNAL-(S)-(+)-α-methoxyl-α-trifluoromethyl phenylacetic acid ester and the standard solution working curve of (R)-NNAL-(S)-(+)-α-methoxyl-α-trifluoromethyl phenylacetic acid ester;
3. the urine sample after at room temperature thawing is shaken up, add rac-NNAL-methyl-d3 solution, biphosphate ammonia buffer (pH6.4, 0.6M) and beta-glucuronidase, fully mixing postposition enters in microwave auxiliary extraction instrument at 37 ℃ of constant temperature enzymolysis, the urine sample after enzymolysis being loaded to MIP-NNAL solid phase extraction column purifies again, after drip washing, use methylene chloride wash-out, after adding anhydrous sodium sulfate drying, filter, be evaporated to dry, add triethylamine anhydrous acetonitrile, (S) anhydrous acetonitrile of-(+)-α-methoxyl-α-trifluoromethyl phenyllacetyl chloride, cover tightly, after derivatization, immediately sample is carried out to GC-MS-MS analysis.
Gas chromatography-mass spectrum-mass spectrometry detection method of (S)-NNAL and (R)-NNAL in described urine, wherein carries out Mosher derivatization when (S)-NNAL and (R)-NNAL in urine in analyzing and testing urine sample.
In described urine, in gas chromatography-mass spectrum-mass spectrometry detection method of (S)-NNAL and (R)-NNAL, use microwave auxiliary extraction instrument, at 37 ℃, urine sample is carried out to constant temperature enzymolysis.
Embodiment
Below in conjunction with Application Example, the present invention is described in further detail.Should be appreciated that specific embodiment described herein, only in order to explain the present invention, does not limit protection scope of the present invention.
Embodiment 1:
Except as otherwise noted, the percentage adopting in the present invention is percent by weight.
Gas chromatography-mass spectrum-mass spectrometry detection method of (S)-NNAL and (R)-NNAL in urine, comprises the following steps:
1. chemical reagent
(S)-(+)-α-methoxyl-α-trifluoromethyl phenyllacetyl chloride (CAS 20445-33-4), (S)-(+)-α--methoxyl-(trifluoromethyl) phenylacetic anhydride (CAS 85541-57-7), 4-(methyl nitrosamino-)-1-(3-pyridine radicals)-butanols (NNAL) (rac NNAL, CAS 76014-81-8), 4-(methyl-d3-nitrosamino-)-1-(3-pyridine radicals)-n-butyl alcohol (rac-NNAL-methyl-d3CAS 1020719-61-2), triethylamine, sweet sour enzyme (the type i X-A of β-glucose, from Escherichia coli)
(S) determining of the preparation of-NNAL and (R)-NNAL and absolute configuration: preparative OD-H chiral chromatographic column, mobile phase isopropyl alcohol: the ratio of normal hexane is 10:90, collect respectively two and flow out chromatographic peak, concentrated until its residue weight is all greater than 1mg respectively.Get respectively the concentrate of two chromatographic peaks, dissolve with 2ml ethanol, measure its specific rotatory power value, the concentrate that specific rotatory power value is negative value is (S)-NNAL, specific rotatory power value be on the occasion of concentrate be (R)-NNAL.
2. typical curve is set up
Raceme NNAL standard solution: take anhydrous acetonitrile as solvent, compound concentration is the rac NNAL standard items list mark solution of 1 μ g/mL, retains four position effective digitals; With the same manner take anhydrous acetonitrile as solvent respectively compound concentration as single mark solution of rac-NNAL-methyl-d3 of 1 μ g/mL; Take anhydrous acetonitrile as solvent, prepare 8 mixed standard solutions that contain rac-NNAL and deuterated rac-NNAL-methyl-d3 by table 1; Compound concentration is the anhydrous acetonitrile of 10ng/mL's (S)-(+)-α-methoxyl-α-trifluoromethyl phenyllacetyl chloride; Compound concentration is the anhydrous acetonitrile of the triethylamine of 10ng/mL; Get respectively 0.5mL8 mixed standard solution in 8 GC-MS-MS chromatogram bottles, under 0 ℃ of degree anhydrous condition, add respectively again anhydrous acetonitrile and the triethylamine acetonitrile solution of (S)-(+)-α-methoxyl-α-trifluoromethyl phenyllacetyl chloride according to table 2, room temperature concussion 10 minutes, at room temperature leaves standstill 2 hours; Then immediately 8 later mixed standard solutions of derivatization are carried out to GC-MS-MS analysis; Drain determining (S)-NNAL or the dissolving of (R)-NNAL acetonitrile after configuration, (S)-(+)-α-methoxyl-α-trifluoromethyl phenyllacetyl chloride carries out derivatization, determines the chromatographic peak of (S)-NNAL-(S)-(+)-α-methoxyl-α-trifluoromethyl phenylacetic acid ester after derivatization and (R)-NNAL-(S)-(+)-α-methoxyl-α-trifluoromethyl phenylacetic acid ester by retention time.The peak area of a pair of diastereo-isomerism that raceme NNAL standard solution derivatization generates is 1:1.In GC-MS-MS chromatogram, the concentration of (S)-NNAL-(S)-(+)-α-methoxyl-α-trifluoromethyl phenylacetic acid ester is horizontal ordinate with the ratio of the concentration of (S)-NNAL-methyl-d3-(S)-(+)-α-methoxyl-α-trifluoromethyl phenylacetic acid ester, in chromatogram, the quota ion of (S)-NNAL-(S)-(+)-α-methoxyl-α-trifluoromethyl phenylacetic acid ester is ordinate with the quota ion of (S)-NNAL-methyl-d3-(S)-(+)-α-methoxyl-α-trifluoromethyl phenylacetic acid ester to the ratio of peak area to peak area, set up the standard solution working curve of (S)-NNAL-(S)-(+)-α-methoxyl-α-trifluoromethyl phenylacetic acid ester, in kind set up the standard solution working curve of (R)-NNAL-(S)-(+)-α-methoxyl-α-trifluoromethyl phenylacetic acid ester.
The mixed standard solution of table 1rac-NNAK and deuterated rac-NNAL-methyl-d3
The derivatization of table 2 mixed standard solution
3. urine sample processing
Urine sample after at room temperature thawing is shaken up, accurately pipette 8mL urine in 50mL test tube, add rac-NNAL-methyl-d3 solution that 100 μ L concentration are 1ng/mL, add again the biphosphate ammonia buffer (pH6.4 of 2.0mL, the beta-glucuronidase (1000U/mL urine) of 0.6M) He 500 μ L, fully mix postposition and enter microwave auxiliary extraction instrument, 37 ℃ of constant temperature enzymolysis 60s, leave standstill 10 minutes, 37 ℃ of constant temperature enzymolysis 60s again, urine sample after enzymolysis is loaded to and uses respectively 1mL methylene chloride, 1mL methyl alcohol, the MIP-NNAL solid phase extraction column that 1mL water activates purifies, with the drip washing of 4mL deionized water and drain, use respectively again 2mL toluene, 1mL toluene/methylene chloride (9:1) wash-out, finally use 3*1mL methylene chloride wash-out, merge eluent, after adding 1.0g anhydrous sodium sulfate drying, filter, be evaporated to dry, adding 0.2mL concentration is the triethylamine anhydrous acetonitrile of 1 μ g/mL, shake 1 minute, under 0 ℃ of degree anhydrous condition, adding 0.2mL concentration is again the anhydrous acetonitrile of 1 μ g/mL (S)-(+)-α-methoxyl-α-trifluoromethyl phenyllacetyl chloride, cover tightly, room temperature concussion 10 minutes, at room temperature leave standstill 2 hours, then immediately sample is carried out to GC-MS-MS analysis.
4.GC-MS-MS testing conditions
DB-5MS capillary column (60m*0.25mm ID*0.25 μ m), using the helium of flow velocity 1mL/min as carrier gas, 270 ℃ of injector temperatures, sample size 2 μ L, do not shunt.
Column temperature: 100 ℃ of initial temperatures, keep 2min, then with 15 ℃/min
-1speed rise to 260 ℃, keep 5min, then with 25 ℃/min
-1speed rise to 320 ℃, keep 3min.250 ℃ of ion source temperatures, ion gun bombarding voltage 70eV.The monitoring parameter that each component GC-MS-MS measures is in table 3.
The each component GC-MS-MS location parameter to be measured of table 3
NNK, (the S)-NNAL relating in said method and the chemical structural formula of (R)-NNAL are:
Embodiment 2:
1. the derivatization of raceme NNAL: take anhydrous acetonitrile as solvent, compound concentration is the rac NNAL standard items list mark solution of 1 μ g/mL, retains four position effective digitals; With the same manner take anhydrous acetonitrile as solvent respectively compound concentration as single mark solution of rac-NNAL-methyl-d3 of 1 μ g/mL; Take anhydrous acetonitrile as solvent, prepare 8 mixed standard solutions that contain rac-NNAL and deuterated rac-NNAL-methyl-d3 by table 1; Compound concentration is the anhydrous acetonitrile of 10ng/mL's (S)-(+)-α-methoxyl-α-trifluoromethyl phenyllacetyl chloride; Compound concentration is the anhydrous acetonitrile of the triethylamine of 10ng/mL; Get respectively 0.5mL8 mixed standard solution in 8 GC-MS-MS chromatogram bottles, under 0 ℃ of degree anhydrous condition, add respectively again anhydrous acetonitrile and the triethylamine acetonitrile solution of (S)-(+)-α-methoxyl-α-trifluoromethyl phenyllacetyl chloride according to table 2, room temperature concussion 10 minutes, at room temperature leaves standstill 2 hours; Then immediately 8 later mixed standard solutions of derivatization are carried out to GC-MS-MS analysis.
2. actual urine sample detection: the urine sample after at room temperature thawing is shaken up, accurately pipette 8mL urine in 50mL test tube, add rac-NNAL-methyl-d3 solution that 100 μ L concentration are 1ng/mL, add again the biphosphate ammonia buffer (pH6.4 of 2.0mL, the beta-glucuronidase (1000U/mL urine) of 0.6M) He 500 μ L, fully mix postposition and enter microwave auxiliary extraction instrument, 37 ℃ of constant temperature enzymolysis 60s, leave standstill 10 minutes, 37 ℃ of constant temperature enzymolysis 60s again, urine sample after enzymolysis is loaded to and uses respectively 1mL methylene chloride, 1mL methyl alcohol, the MIP-NNAL solid phase extraction column that 1mL water activates purifies, with the drip washing of 4mL deionized water and drain, use respectively again 2mL toluene, 1mL toluene/methylene chloride (9:1) wash-out, finally use 3*1mL methylene chloride wash-out, merge eluent, after adding 1.0g anhydrous sodium sulfate drying, filter, be evaporated to dry, adding 0.2mL concentration is the triethylamine anhydrous acetonitrile of 1 μ g/mL, shake 1 minute, under 0 ℃ of degree anhydrous condition, adding 0.2mL concentration is again the anhydrous acetonitrile of 1 μ g/mL (S)-(+)-α-methoxyl-α-trifluoromethyl phenyllacetyl chloride, cover tightly, room temperature concussion 10 minutes, at room temperature leave standstill 2 hours, then immediately sample is carried out to GC-MS-MS analysis.Use the standard solution working curve of (S)-NNAL-(S)-(+)-α-methoxyl-α-trifluoromethyl phenylacetic acid ester and the standard solution working curve of (R)-NNAL-(S)-(+)-α-methoxyl-α-trifluoromethyl phenylacetic acid ester to calculate (S)-NNAL-(S)-(+)-α-methoxyl-α-trifluoromethyl phenylacetic acid ester and (R)-NNAL-(S)-(+)-α-methoxyl-α-trifluoromethyl phenylacetic acid ester in testing sample, and then converse the concentration of (S)-NNAL and (R)-NNAL in this urine and be respectively 0.96ng/mL and 0.83ng/mL.
Embodiment 3:
1. determining of the preparation of (S)-NNAL and (R)-NNAL and absolute configuration: carry out respectively Mosher reaction with anhydrous acetonitrile, the anhydrous acetonitrile of (S)-(+)-α-methoxyl-α-trifluoromethyl phenylacetic acid acid anhydride and the anhydrous acetonitrile of (R)-(+)-α-methoxyl-α-trifluoromethyl phenylacetic acid acid anhydride and the triethylamine of rac NNAL standard items, then pass through purifying with
1the test of H NMR spectrogram, by the absolute configuration of definite (S)-NNAL-(S)-(+)-α-methoxyl-α-trifluoromethyl phenylacetic acid ester of Mosher method and (R)-NNAL-(S)-(+)-α-methoxyl-α-trifluoromethyl phenylacetic acid ester.
2. the derivatization of raceme NNAL and the foundation of typical curve: take anhydrous acetonitrile as solvent, compound concentration is the rac NNAL standard items list mark solution of 1 μ g/mL, retains four position effective digitals; With the same manner take anhydrous acetonitrile as solvent respectively compound concentration as single mark solution of rac-NNAL-methyl-d3 of 1 μ g/mL; Take anhydrous acetonitrile as solvent, prepare 8 mixed standard solutions that contain rac-NNAL and deuterated rac-NNAL-methyl-d3 by table 1; Compound concentration is the anhydrous acetonitrile of 10ng/mL's (S)-(+)-α-methoxyl-α-trifluoromethyl phenylacetic acid acid anhydride; Compound concentration is the anhydrous acetonitrile of the triethylamine of 10ng/mL; Get respectively 0.5mL8 mixed standard solution in 8 GC-MS-MS chromatogram bottles, under 0 ℃ of degree anhydrous condition, add respectively again anhydrous acetonitrile and the triethylamine acetonitrile solution of (S)-(+)-α-methoxyl-α-trifluoromethyl phenylacetic acid acid anhydride, room temperature concussion 10 minutes, at room temperature leaves standstill 2 hours; Then immediately 8 later mixed standard solutions of derivatization are carried out to GC-MS-MS analysis.With determining that (S)-NNAL-(S)-(+)-α-methoxyl-α-trifluoromethyl phenylacetic acid ester after configuration and (R)-NNAL-(S)-(+)-α-methoxyl-α-trifluoromethyl phenylacetic acid ester are as reference, determine (S)-NNAL-(S)-(+)-α-methoxyl-α-trifluoromethyl phenylacetic acid ester in the mixed standard solution after derivatization and the chromatographic peak of (R)-NNAL-(S)-(+)-α-methoxyl-α-trifluoromethyl phenylacetic acid ester by retention time.The peak area of a pair of diastereo-isomerism that raceme NNAL standard solution derivatization generates is 1:1.In GC-MS-MS chromatogram, the concentration of (S)-NNAL-(S)-(+)-α-methoxyl-α-trifluoromethyl phenylacetic acid ester is horizontal ordinate with the ratio of the concentration of (S)-NNAL-methyl-d3-(S)-(+)-α-methoxyl-α-trifluoromethyl phenylacetic acid ester, in chromatogram, the quota ion of (S)-NNAL-(S)-(+)-α-methoxyl-α-trifluoromethyl phenylacetic acid ester is ordinate with the quota ion of (S)-NNAL-methyl-d3-(S)-(+)-α-methoxyl-α-trifluoromethyl phenylacetic acid ester to the ratio of peak area to peak area, set up the standard solution working curve of (S)-NNAL-(S)-(+)-α-methoxyl-α-trifluoromethyl phenylacetic acid ester, in kind set up the standard solution working curve of (R)-NNAL-(S)-(+)-α-methoxyl-α-trifluoromethyl phenylacetic acid ester.
3. actual urine sample detection: the urine sample after at room temperature thawing is shaken up, accurately pipette 8mL urine in 50mL test tube, add rac-NNAL-methyl-d3 solution that 100 μ L concentration are 1ng/mL, add again the biphosphate ammonia buffer (pH6.4 of 2.0mL, the beta-glucuronidase (1000U/mL urine) of 0.6M) He 500 μ L, fully mix postposition and enter microwave auxiliary extraction instrument, 37 ℃ of constant temperature enzymolysis 60s, leave standstill 10 minutes, 37 ℃ of constant temperature enzymolysis 60s again, urine sample after enzymolysis is loaded to and uses respectively 1mL methylene chloride, 1mL methyl alcohol, the MIP-NNAL solid phase extraction column that 1mL water activates purifies, with the drip washing of 4mL deionized water and drain, use respectively again 2mL toluene, 1mL toluene/methylene chloride (9:1) wash-out, finally use 3*1mL methylene chloride wash-out, merge eluent, after adding 1.0g anhydrous sodium sulfate drying, filter, be evaporated to dry, adding 0.2mL concentration is the triethylamine anhydrous acetonitrile of 1 μ g/mL, shake 1 minute, under 0 ℃ of degree anhydrous condition, adding 0.2mL concentration is again the anhydrous acetonitrile of 1 μ g/mL (S)-(+)-α-methoxyl-α-trifluoromethyl phenylacetic acid acid anhydride, cover tightly, room temperature concussion 10 minutes, at room temperature leave standstill 2 hours, then immediately sample is carried out to GC-MS-MS analysis.Use the standard solution working curve of (S)-NNAL-(S)-(+)-α-methoxyl-α-trifluoromethyl phenylacetic acid ester and the standard solution working curve of (R)-NNAL-(S)-(+)-α-methoxyl-α-trifluoromethyl phenylacetic acid ester to calculate (S)-NNAL-(S)-(+)-α-methoxyl-α-trifluoromethyl phenylacetic acid ester and (R)-NNAL-(S)-(+)-α-methoxyl-α-trifluoromethyl phenylacetic acid ester in testing sample, and then converse the concentration of (S)-NNAL and (R)-NNAL in this urine and be respectively 0.61ng/mL and 0.53ng/mL.
Embodiment 4:
1. the derivatization of raceme NNAL: take anhydrous acetonitrile as solvent, compound concentration is the rac NNAL standard items list mark solution of 1 μ g/mL, retains four position effective digitals; With the same manner take anhydrous acetonitrile as solvent respectively compound concentration as single mark solution of rac-NNAL-methyl-d3 of 1 μ g/mL; Take anhydrous acetonitrile as solvent, prepare 8 mixed standard solutions that contain rac-NNAL and deuterated rac-NNAL-methyl-d3 by table 1; Compound concentration is the anhydrous acetonitrile of 10ng/mL's (S)-(+)-α-methoxyl-α-trifluoromethyl phenyllacetyl chloride; Compound concentration is the anhydrous acetonitrile of the triethylamine of 10ng/mL; Get respectively 0.5mL8 mixed standard solution in 8 GC-MS-MS chromatogram bottles, under 0 ℃ of degree anhydrous condition, add respectively again anhydrous acetonitrile and the triethylamine acetonitrile solution of (S)-(+)-α-methoxyl-α-trifluoromethyl phenyllacetyl chloride according to table 2, room temperature concussion 10 minutes, at room temperature leaves standstill 2 hours; Then immediately 8 later mixed standard solutions of derivatization are carried out to GC-MS-MS analysis.
2. actual urine sample detection: the urine sample after at room temperature thawing is shaken up, accurately pipette 8mL urine in 50mL test tube, add rac-NNAL-methyl-d3 solution that 100 μ L concentration are 1ng/mL, add again the biphosphate ammonia buffer (pH6.4 of 2.0mL, the beta-glucuronidase (1000U/mL urine) of 0.6M) He 500 μ L, after fully mixing in 37 ℃ of waters bath with thermostatic control enzymolysis 16 hours, urine sample after enzymolysis is loaded to and uses respectively 1mL methylene chloride, 1mL methyl alcohol, the MIP-NNAL solid phase extraction column that 1mL water activates purifies, with the drip washing of 4mL deionized water and drain, use respectively again 2mL toluene, 1mL toluene/methylene chloride (9:1) wash-out, finally use 3*1mL methylene chloride wash-out, merge eluent, after adding 1.0g anhydrous sodium sulfate drying, filter, be evaporated to dry, adding 0.2mL concentration is the triethylamine anhydrous acetonitrile of 1 μ g/mL, shake 1 minute, under 0 ℃ of degree anhydrous condition, adding 0.2mL concentration is again the anhydrous acetonitrile of 1 μ g/mL (S)-(+)-α-methoxyl-α-trifluoromethyl phenyllacetyl chloride, cover tightly, room temperature concussion 10 minutes, at room temperature leave standstill 2 hours, then immediately sample is carried out to GC-MS-MS analysis.Use the standard solution working curve of (S)-NNAL-(S)-(+)-α-methoxyl-α-trifluoromethyl phenylacetic acid ester and the standard solution working curve of (R)-NNAL-(S)-(+)-α-methoxyl-α-trifluoromethyl phenylacetic acid ester to calculate (S)-NNAL-(S)-(+)-α-methoxyl-α-trifluoromethyl phenylacetic acid ester and (R)-NNAL-(S)-(+)-α-methoxyl-α-trifluoromethyl phenylacetic acid ester in testing sample, and then converse the concentration of (S)-NNAL and (R)-NNAL in this urine and be respectively 1.16ng/mL and 1.13ng/mL.
It may be noted that and the foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any modifications of doing within the spirit and principles in the present invention, be equal to and replace and improvement etc., within all should being included in protection scope of the present invention.
Compared with prior art, the present invention has following outstanding advantages:
1. the present invention uses derivatization-GC-MS-MS method to measure (S)-NNAL and (R)-NNAL in urine, have that detectability is low, good stability, pre-treatment simply, the advantage such as quick and precisely;
2. affected by subjective factor of judgment little for the testing result of the inventive method, is easy to apply.
3, be difficult to carry out the deficiency of the testing of (S)-NNAL and (R)-NNAL in urine sample for existing method, provide the gas chromatography-mass spectrum-mass spectrometry detection method of (S)-NNAL and (R)-NNAL in a kind of urine, for assessing the individual decision method that the degree of exposure of NNK in flue gas is provided to a kind of science.
4, gas chromatography-mass spectrum-mass spectrometry that this method is applicable to (S)-NNAL and (R)-NNAL in urine detects, and is the method for the degree of exposure of a kind of Scientific evaluation individuality to NNK in flue gas.
Claims (1)
1. gas chromatography-mass spectrum-mass spectrometry detection method of (S)-NNAL and (R)-NNAL in urine, is characterized in that the method comprises the following steps:
1. with the configuration of mensuration optically-active definite (the S)-NNAL of method comparing and (R)-NNAL and as reference, or with the configuration of definite (S)-NNAL-(S)-(+)-α-methoxyl-α-trifluoromethyl phenylacetic acid ester of Mosher method and (R)-NNAL-methyl-d3-(S)-(+)-α-methoxyl-α-trifluoromethyl phenylacetic acid ester and as reference;
2. the mixed standard solution that preparation contains rac-NNAL and deuterated rac-NNAL-methyl-d3; use again the anhydrous acetonitrile of (S)-(+)-α-methoxyl-α-trifluoromethyl phenyllacetyl chloride and the anhydrous acetonitrile derivatization of triethylamine, then the later mixed standard solution of derivatization is carried out to GC-MS-MS analysis; With determining that (S)-NNAL-(S)-(+)-α-methoxyl-α-trifluoromethyl phenylacetic acid ester and (R)-NNAL-(S)-(+)-α-methoxyl-α-trifluoromethyl phenylacetic acid ester after configuration determine corresponding chromatographic peak retention time by retention time, and set up the standard solution working curve of (S)-NNAL-(S)-(+)-α-methoxyl-α-trifluoromethyl phenylacetic acid ester and the standard solution working curve of (R)-NNAL-(S)-(+)-α-methoxyl-α-trifluoromethyl phenylacetic acid ester;
3. the urine sample after at room temperature thawing is shaken up, add rac-NNAL-methyl-d3 solution, biphosphate ammonia buffer and beta-glucuronidase, fully mixing postposition enters in microwave auxiliary extraction instrument at 37 ℃ of constant temperature enzymolysis, the urine sample after enzymolysis being loaded to MIP-NNAL solid phase extraction column purifies again, after drip washing, use methylene chloride wash-out, after adding anhydrous sodium sulfate drying, filter, be evaporated to dry, add triethylamine anhydrous acetonitrile, (S) anhydrous acetonitrile of-(+)-α-methoxyl-α-trifluoromethyl phenyllacetyl chloride, cover tightly, after derivatization, immediately sample is carried out to GC-MS-MS analysis, chromatographic column adopting DB-5MS capillary column, 60m*0.25mm ID*0.25 μ m, heating schedule is: column temperature: 100 ℃ of initial temperatures, keep 2min, and then rise to 260 ℃ with the speed of 15 ℃/min, keep 5min, then rise to 320 ℃ with the speed of 25 ℃/min, keep 3min.
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